EFFECT OF NA-FREE CA-ATPASE FROM SCALLOP SARCOPLASMIC-RETICULUM( AND NUCLEOTIDE ON THE STABILITY OF SOLUBILIZED CA2+)

In membranous scallop sarcoplasmic reticulum, the alkali metal cations Na+ and K+ and nucleotide together promote dimer formation by the Ca2 +-free Ca-ATPase and stabilize the enzyme activity [Kalabokis, V. N., Bozzola, J. J., Castellani, L., & Hardwicke, P. M. D. (I 99 1) J. Biol . Chem. 266, 22044-22050]. The dependence of stabilization of the Ca2-free membranous scallop Ca-ATPase on Na+ concentration does not show saturation and may involve several superimposed effects. In order to a ssess the contribution of dimer toward stabilization, i.e., determine the relative importance of intra- and intermolecular effects on stabil ization, the influence of varying Na+ concentration and nucleotide on the decay of enzyme activity of the Ca2+-free detergent-solubilized Ca -ATPase was studied. Loss of enzyme activity on removal of Ca2+ with E GTA was associated with loss of capacity for phosphorylation by ATP, a Ca2+-dependent function. Stabilization of the soluble Ca2+-free enzym e by Na+ showed major differences from that seen with the membranous e nzyme. The extent of stabilization of the Ca2+-free soluble enzyme by Na+ showed clear saturation with increasing Na+ concentration. In cont rast to the Ca2+-free membranous enzyme, which is inactivated at pH 7. 0 with biphasic first-order kinetics, loss of enzymatic function by th e solubilized Ca-ATPase at pH 6.92, 0-degrees-C, followed monophasic f irst-order kinetics. Investigation of the aggregational state of the N a+-stabilized, Ca2+-free soluble enzyme by gel permeation chromatograp hy showed that it was monomeric, and this may be related to the differ ences between the effects of Na+ on the membranous and soluble systems , since nonsolubilizing levels of C12E8 (below the critical micelle co ncentration) did not affect the decay of the enzyme activity of the me mbranous scallop Ca-ATPase observed in the absence of detergent (Kalab okis et al., 1991). By carrying out the incubations at constant ionic strength, it was found that the slowing of the inactivation rate of th e Ca2+-free soluble scallop Ca-ATPase by Na+ was not due to purely ion ic strength effects, but was consistent with a simple kinetic model in which binding of Na+ to a saturable, weak binding site on the Ca2+-fr ee scallop Ca-ATPase of K(d) almost-equal-to 3 mM mediates stabilizati on by the ion. AMP-PCP decreased the rate constant for inactivation of the Na+-free, Ca2+-free Ca-ATPase, but did not affect the decay of th e Na+-liganded form or the affinity of the Ca2+-free enzyme for Na+; i .e., Na+ and nucleotide act independently. Nucleotide was far less eff ective in stabilizing the Ca2+-free solubilized enzyme than the Ca2+-f ree membranous scallop Ca-ATPase, and this is probably explained by th e greater opportunity for intermolecular contacts, and therefore dimer formation, by Ca-ATPase located in the SR membrane.